CN113069554A - Preparation method and application of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles - Google Patents
Preparation method and application of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles Download PDFInfo
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Abstract
The invention relates to a preparation method and application of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles, which effectively solve the preparation of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles, realize the application in the preparation of anti-inflammatory, antihypertensive, anticancer and antibacterial drugs, improve the drug absorption rate and curative effect, dissolve oleanolic acid with solvent, addIntoN,N-diisopropylethylamine, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 1-hydroxybenzotriazole, ultrasonic reacting, addingN,NAnd (2) performing amidation reaction on dimethyl ethylenediamine to obtain an intermediate compound, dissolving the intermediate compound in an organic solvent, adding inorganic base and methyl iodide to obtain oleanolic acid quaternary ammonium salt, dissolving the oleanolic acid quaternary ammonium salt in methanol, adding a heparin deionized water solution, stirring overnight, evaporating to obtain a film, hydrating the film, performing probe ultrasonic treatment, centrifuging, filtering supernate to obtain an oleanolic acid quaternary ammonium salt-heparin nanoparticle solution, dropwise adding the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution into a chitosan acetic acid solution, centrifuging, and filtering the supernate to obtain the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticle.
Description
Technical Field
The invention relates to medicine, in particular to a preparation method and application of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles.
Background
Oleanolic Acid (OA) is an oleanane type pentacyclic triterpene compound, is a natural plant extract, is widely distributed in nature in free form or combined with sugar into glycoside form, and exists in leaves of Olea europaea L of Oleaceae and Ligustrum lucidum ait fruit; swertia mussotii Franch of Gentianaceae family, and swertia mileensis Franch; leaf and root of Oenanthe stolonifera of Umbelliferae; root bark and stem bark of Demu of Araliaceae; root tuber of Cucurbitaceae plant such as semen Strychni, Aixuedai, and Chinese Hemsleya (radix Hemsleyae Macrospermae ). Has effects in protecting liver, relieving inflammation, resisting virus, resisting oxidation, and regulating immunity. OA has low water solubility and poor oral absorption, but in view of its definite pharmacological actions, researchers at home and abroad have made a lot of research work on its modification. OA is used as a lead compound to synthesize derivatives of OA, and the obtained derivatives are researched to screen and explore substances with larger bioactivity potential. The pentacyclic triterpene skeleton of the oleanolic acid has high rigidity, and meanwhile, the structure lacks a water-soluble segment, so that the oleanolic acid has poor water solubility, and the poor water solubility directly influences the bioavailability and the bioactivity. The nano technology can improve the solubility, stability, bioavailability and the like of the medicine, and researches show that the oleanolic acid derivative preparation can play the anti-breast cancer activity by regulating intracellular mitochondrial signaling pathways. Heparin (HEP) is a polyanionic polysaccharide that exhibits electronegativity at physiological pH, and sulfonic and carboxyl groups in linear sugar chains can bind positively charged compounds through electrostatic interaction, such as Heparin in complex with positively charged berberine or doxorubicin. Chitosan (Chitosan, CS) is a natural material with good biocompatibility with mammalian cells, and consists of cationic glucosamine residues constituting polysaccharide chains. Hydrophobic drugs are introduced into the heparin or chitosan nanoparticles, so that the low water-solubility drugs can be ensured to play an anti-tumor role. Aiming at the problems of poor water solubility, low bioavailability and the like of oleanolic acid, the effective treatment of tumors is difficult to realize. A prodrug delivery system based on polycations and anionic carriers can be used to increase the solubility of poorly soluble drugs. Therefore, a prodrug of oleanolic acid is designed and synthesized, heparin and chitosan are used as carriers of the prodrug to construct nano particles, and improvement of absorption efficiency and improvement of antitumor effect of the nano particles are very necessary, but no public report is found so far.
Disclosure of Invention
In view of the above situation, the present invention aims to provide a preparation method and an application of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs), which can effectively solve the problems of preparation of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles, application in preparing anti-inflammatory, antihypertensive, anticancer and antibacterial drugs, and improvement of drug absorption rate and curative effect.
The technical scheme of the invention is that the preparation method of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs) comprises the following steps:
(1) preparation of intermediate compound (QDN):
first, Oleanolic Acid (OA) is dissolved in an organic solvent, and then addedN,NDiisopropylethylamine (N-ethylisopropylamine, DIPEA for short), 1-ethyl-3 (3-dimethylpropylamine) carbodiimide (1-ethyl-3- (3-methylenepropyl carbodiimide hydrochloride), EDCI for short) and 1-Hydroxybenzotriazole (1-Hydroxybenzotriazole, HOBT for short), ultrasonically assisted to dissolve so as to enable the solution to be clear, and stirring and reacting for 8-12 hours at room temperature or in ice bath; then is added toN,N-dimethylethylenediamine(N,NDimethyl ethylene diamine) to perform amidation reaction at 0-25 ℃ for 12-24 hours under stirring, wherein the molar ratio of oleanolic acid to 1-ethyl-3 (3-dimethylpropylamine) carbodiimide to 1-hydroxybenzotriazoleN,NDiisopropylethylamine (diisopropylethylamine)N,N-dimethylethylenediamine (QDN) at a ratio of 1: 1.2-3 to obtain Oleanolic acid amidation intermediate compound (QDN);
the organic solvent is one or a mixture of more than two of tetrahydrofuran, dichloromethane, trichloromethane, acetone and methanol in any volume ratio;
(2) preparation of oleanolic acid quaternary ammonium salt (QDT):
dissolving the intermediate compound (QDN) in an organic solvent, and adding dried inorganic base and methyl iodide (CH)3I) The molar ratio of the intermediate compound to the inorganic base to the methyl iodide is 1: 1.5: 3-5, the reaction temperature is 50-100 ℃, and the Oleanolic acid quaternary ammonium salt (QDT) is obtained by stirring and reacting for 4-12 hours in a dark place;
the inorganic base is one of potassium carbonate, sodium carbonate, cesium carbonate and sodium bicarbonate;
(3) and preparing an oleanolic acid quaternary ammonium salt-heparin nanoparticle (QDT-HEP NPs) solution:
respectively dissolving oleanolic acid quaternary ammonium salt (QDT) and Heparin (HEP) in methanol and deionized water, dripping the oleanolic acid quaternary ammonium salt solution into the heparin solution at room temperature, wherein the mass ratio of the oleanolic acid quaternary ammonium salt (QDT) to the Heparin (HEP) is 4-12: 4-12, stirring for 8-12h overnight, performing reduced pressure rotary evaporation at 37 ℃ to obtain a film, hydrating the film with deionized water, performing ultrasonic treatment on a probe for 5min, centrifuging at 10000rpm for 10min, taking supernatant, and filtering with a filter membrane with the aperture of 0.45 mu m to obtain an oleanolic acid quaternary ammonium salt-heparin nanoparticle (QDT-HEP) solution;
(4) and preparing oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs):
dripping the solution of oleanolic acid quaternary ammonium salt-heparin nanoparticles (QDT-HEP NPs) into a Chitosan (CS) acetic acid solution, stirring for 2h, centrifuging at 10000rpm for 10min, taking supernatant, filtering with a filter membrane with the pore diameter of 0.45 mu m to obtain polyelectrolyte nanoparticle solution with surface modified chitosan, concentrating and drying to obtain the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs);
the pH value of the chitosan acetic acid solution is 6.0.
The oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs) have the effects of inducing apoptosis of cancer cells and inhibiting activity of tumor cells, and can be effectively used for preparing anti-inflammatory, antihypertensive, anticancer and antibacterial medicaments.
The method is novel and unique, is easy to operate, is efficient and controllable, has good product stability, develops a new way for preparing the anti-inflammatory, antihypertensive, anticancer and antibacterial drugs, is a great innovation on the drugs, and has remarkable economic and social benefits.
Drawings
FIG. 1 is a graph showing the comparison of in vitro stability of the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles of the present invention.
FIG. 2 is a comparative graph of toxicity tests of the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles and free oleanolic acid quaternary ammonium salt (QDT) on 4T1 cells.
FIG. 3 is a graph of the tumor volume change measured (a) according to the present invention (b) the change in body weight of mice during treatment (c) the mean tumor weight (d) the tumor inhibition rate.
FIG. 4 is a graph showing hydrogen spectrum data of an oleanolic acid amidated intermediate compound of the present invention.
FIG. 5 is a graph of hydrogen spectrum data of quaternary ammonium oleanolic acid salt (QDT) of the present invention.
Detailed Description
The following examples and specific examples will explain the present invention in detail.
In particular, the invention may be embodied as set forth in the following examples.
Example 1
The invention relates to a preparation method of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs), which comprises the following steps:
(1) preparation of intermediate compound (QDN):
firstly, dissolving oleanolic acid in organic solvent tetrahydrofuran, and then addingN,N-Diisopropylethylamine, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 1-hydroxybenzotriazole, and ultrasonic-assisted dissolution is carried out to clarify the solution, and the solution is stirred and reacts for 10 hours at room temperature; then is added toN,NDimethyl ethylenediamine, amidating at 12 deg.c under stirring for 18 hr, and oleanolic acid/1-ethyl-3 (3-dimethylpropylamine) carbodiimide/1-hydroxybenzotriazole/N,NDiisopropylethylamine (diisopropylethylamine)N,N-dimethylethylenediamine (QDN) at a ratio of 1: 2 to obtain oleanolic acid amidation intermediate compound (QDN);
(2) preparation of oleanolic acid quaternary ammonium salt (QDT):
dissolving intermediate compound (QDN) in organic solvent tetrahydrofuran, and adding dried inorganic base potassium carbonate and iodomethane (CH)3I) The molar ratio of the intermediate compound to potassium carbonate to methyl iodide is 1: 1.5: 4, the reaction temperature is 75 ℃, and the oleanolic acid quaternary ammonium salt (QDT) is obtained after the reaction is stirred for 4 to 12 hours in a dark place;
(3) and preparing an oleanolic acid quaternary ammonium salt-heparin nanoparticle (QDT-HEP NPs) solution:
dissolving oleanolic acid quaternary ammonium salt (QDT) and Heparin (HEP) in methanol and deionized water respectively, dripping the oleanolic acid quaternary ammonium salt solution into the heparin solution at room temperature, wherein the mass ratio of the oleanolic acid quaternary ammonium salt (QDT) to the Heparin (HEP) is 8: 8, stirring overnight for 10h, performing reduced pressure rotary evaporation at 37 ℃ to form a film, hydrating the film with deionized water, performing probe ultrasonic treatment for 5min, centrifuging at 10000rpm for 10min, taking supernatant, and filtering with a filter membrane with the aperture of 0.45 mu m to obtain an oleanolic acid quaternary ammonium salt-heparin nanoparticle (QDT-HEPNNPs) solution;
(4) and preparing oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs):
dripping solution of oleanolic acid quaternary ammonium salt-heparin nanoparticles (QDT-HEP NPs) into Chitosan (CS) acetic acid solution, stirring for 2h, centrifuging at 10000rpm for 10min, collecting supernatant, filtering with 0.45 μm pore size filter membrane to obtain polyelectrolyte nanoparticle solution with surface modified chitosan, concentrating, and drying to obtain oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs).
Example 2
The invention relates to a preparation method of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs), which comprises the following steps:
(1) preparation of intermediate compound (QDN):
first, Oleanolic Acid (OA) is dissolved in organic solvent methanol, and then addedN,NDiisopropylethylamine, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 1-hydroxybenzotriazole, and ultrasonic-assisted dissolution is carried out to clarify the solution, and the solution is stirred in an ice bath and reacts for 11 hours; then is added toN,NDimethyl ethylenediamine, amidating at 10 deg.c for 20 hr while stirring, and oleanolic acid/1-ethyl-3 (3-dimethylpropylamine) carbodiimide/1-hydroxybenzotriazole/N,NDiisopropylethylamine (diisopropylethylamine)N,N-dimethylethylenediamine (QDN) at 1: 1.3 to obtain Oleanolic acid amidation intermediate compound (QDN);
(2) preparation of oleanolic acid quaternary ammonium salt (QDT):
dissolving the intermediate compound (QDN) in an organic solvent methanol, and adding dried inorganic bases cesium carbonate and iodomethane (CH)3I) The molar ratio of the intermediate compound to cesium carbonate to methyl iodide is 1: 1.5: 3, the reaction temperature is 60 ℃, and the Oleanolic acid quaternary ammonium salt (QDT) is obtained after the reaction is stirred for 11 hours in the dark;
(3) and preparing an oleanolic acid quaternary ammonium salt-heparin nanoparticle (QDT-HEP NPs) solution:
dissolving oleanolic acid quaternary ammonium salt (QDT) and Heparin (HEP) in methanol and deionized water respectively, dripping the oleanolic acid quaternary ammonium salt solution into the heparin solution at room temperature, wherein the mass ratio of the oleanolic acid quaternary ammonium salt (QDT) to the Heparin (HEP) is 4.5: 4, stirring overnight for 8h, performing reduced pressure rotary evaporation at 37 ℃ to obtain a film, hydrating the film with deionized water, performing ultrasonic treatment on a probe for 5min, centrifuging at 10000rpm for 10min, taking supernatant, and filtering with a filter membrane with the aperture of 0.45 mu m to obtain an oleanolic acid quaternary ammonium salt-heparin nanoparticle (QDT-HEP NPs) solution;
(4) and preparing oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs):
dripping solution of oleanolic acid quaternary ammonium salt-heparin nanoparticles (QDT-HEP NPs) into Chitosan (CS) acetic acid solution, stirring for 2h, centrifuging at 10000rpm for 10min, collecting supernatant, filtering with 0.45 μm pore size filter membrane to obtain polyelectrolyte nanoparticle solution with surface modified chitosan, concentrating, and drying to obtain oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs).
Example 3
The invention relates to a preparation method of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs), which comprises the following steps:
(1) preparation of intermediate compound (QDN):
first, Oleanolic Acid (OA) is dissolved in an organic solvent, and then addedN,NDiisopropylethylamine, 1-ethyl-3 (3-dimethylpropan)Amine) carbodiimide and 1-hydroxybenzotriazole, and ultrasonic-assisted dissolution is carried out to clarify the solution, and the solution is stirred and reacts for 9 hours at room temperature; then is added toN,NDimethyl ethylenediamine, amidating at 23 deg.c with stirring for 13 hr, and oleanolic acid/1-ethyl-3 (3-dimethylpropylamine) carbodiimide/1-hydroxybenzotriazole/N,NDiisopropylethylamine (diisopropylethylamine)N,N-dimethylethylenediamine (QDN) at a ratio of 1: 2.9 to obtain oleanolic acid amidation intermediate compound (QDN);
the organic solvent is a mixture of dichloromethane: trichloromethane = 1:1 in volume ratio;
(2) preparation of oleanolic acid quaternary ammonium salt (QDT):
dissolving the intermediate compound (QDN) in an organic solvent, and adding dried inorganic bases sodium bicarbonate and methyl iodide (CH)3I) The molar ratio of the intermediate compound to the sodium bicarbonate to the methyl iodide is 1: 1.5: 5, the reaction temperature is 90 ℃, and the oleanolic acid quaternary ammonium salt (QDT) is obtained after 5 hours of stirring reaction in the dark;
(3) and preparing an oleanolic acid quaternary ammonium salt-heparin nanoparticle (QDT-HEP NPs) solution:
dissolving oleanolic acid quaternary ammonium salt (QDT) and Heparin (HEP) in methanol and deionized water respectively, dripping the oleanolic acid quaternary ammonium salt solution into the heparin solution at room temperature, wherein the mass ratio of the oleanolic acid quaternary ammonium salt (QDT) to the Heparin (HEP) is 6: 8, stirring overnight for 12h, performing reduced pressure rotary evaporation at 37 ℃ to form a film, hydrating the film with deionized water, performing probe ultrasonic treatment for 5min, centrifuging at 10000rpm for 10min, taking supernatant, and filtering with a filter membrane with the aperture of 0.45 mu m to obtain an oleanolic acid quaternary ammonium salt-heparin nanoparticle (QDT-HEPNPs) solution;
(4) and preparing oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs):
dripping solution of oleanolic acid quaternary ammonium salt-heparin nanoparticles (QDT-HEP NPs) into Chitosan (CS) acetic acid solution, stirring for 2h, centrifuging at 10000rpm for 10min, collecting supernatant, filtering with 0.45 μm pore size filter membrane to obtain polyelectrolyte nanoparticle solution with surface modified chitosan, concentrating, and drying to obtain oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs).
Example 4
The invention relates to a preparation method of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs), wherein an intermediate compound (QDN) is prepared by weighing 200mg (0.4 mmol) of oleanolic acid, 180mg (1.3 mmol) of HOBT and 250mg (1.3 mmol) of EDCI into a single-mouth bottle, and dropwise adding 200 mu L (1.3 mmol) of DIPEA and CH2Cl25mL of the solution is ultrasonically dissolved, and the solution is stirred for 8 hours in ice-water bath; thin Layer Chromatography (TLC) monitored the reaction progress, developing agent: dichloromethane: methanol: 15: 1, developer: sulfuric acid-ethanol solution with mass concentration of 10 percent is dripped when the raw material point disappearsN,N-130 mu L (1.2 mmol) of dimethyl ethylenediamine reacts at room temperature, the reaction is stopped when an activation point disappears, the solvent is removed by reduced pressure rotary evaporation, the crude product is dissolved by ethyl acetate and extracted by an isometric saturated NaCl aqueous solution for 3 times, a white oily substance is obtained by concentration of a rotary evaporator, the white oily substance is separated and purified by a flash chromatography system by using petroleum ether/ethyl acetate (volume ratio) 5: 1, the solvent is removed by evaporation of a centrifugal concentrator, and the white solid Oleanolic acid amidated intermediate compound (QDN) is obtained by vacuum drying, 101.5mg and the yield is 43.9%.
Example 5
The invention relates to a preparation method of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles (QDT-HEP-CS NPs), wherein the preparation method of oleanolic acid quaternary ammonium salt (QDT) comprises the steps of precisely weighing 100mg (0.19 mmol) of oleanolic acid amidated intermediate compound (QDN), dissolving the intermediate compound with 2mL of MeOH, adding K2CO331.5mg (0.22 mmol), followed by dropwise addition of 35.5. mu.L (0.57 mmol) of methyl iodide, reflux reaction at 70 ℃ and monitoring of the progress of the reaction by thin layer chromatography, developing agent: dichloromethane: methanol 5: 1, developer: stopping the reaction when the mass concentration of the sulfuric acid-ethanol is 10 percent and the raw material point disappears; filtering the reaction solution, and removing the solvent by reduced pressure evaporation to obtain a white oily substance; separating and purifying with acetonitrile/pure water 90: 10, volatilizing solvent in a test tube concentrator, recrystallizing with diethyl ether, and vacuum drying to obtain white solidThe quaternary ammonium salt of Oleanolic acid (QDT), 112.7mg, 88.7% yield.
The mass ratio of the oleanolic acid quaternary ammonium salt (QDT) to the Heparin (HEP) in the step (3) is also 4.5: 6, 4.5: 8, 4.5: 10 and 4.5: 12; 4: 8, 10: 8 or 12: 8;
the mass ratio of the oleanolic acid quaternary ammonium salt-heparin nanoparticles to the chitosan in the step (4) is also 16: 0.7 or 16: 0.8.
It should be noted that the above-mentioned embodiments are only for clearly illustrating the specific embodiments of the present invention, and do not limit the protection scope of the technical solution claimed in the present invention. For those skilled in the art, on the basis of the foregoing description, other variations or modifications may be made, and all the modifications or modifications made are the same as the present invention in nature and fall within the scope of the present invention, and all the embodiments need not be exhaustive.
The products of the embodiments 1-3 of the invention are identified by a nuclear magnetic resonance spectrometer, are the same products, and have obvious anti-tumor effect on mouse breast cancer 4T1 cells, thereby improving the curative effect of oleanolic acid on breast cancer, and the related data are as follows:
1. structural characterization of oleanolic acid amidated intermediate compound (QDN): c34H58N2O2，1H-NMR (500 MHz, CDCl3) And (3) spectrum display:δ H 5.38 (1H, br.s, H-12) is a hydrogen proton on a double bond,δ H3.23 (1H, t, J =2.7 Hz, H-3) is hydrogen on an oxocarbon,δ H7 methyl hydrogen protons appear between 0.75 and 1.2,δ H 2.58 (1H, dd, J=14.1, 5 Hz, H-18)，δ Ha plurality of CH and CH2 hydrogen signals appear between 1.0 and 2.1,δ H 3.38 (2H, t, J = 11.4, 4.3 Hz), δ H2.54 to 2.45 (t, 2H) is a hydrogen proton on the side chain of ethylenediamine,δ H2.33 (s, 6H) is a methyl hydrogen proton on the nitrogen atom.
2. Oleanolic acid quaternary ammonium salt (QDT)Structural characterization of (a): c35H61N2O2I, molecular weight of 668.3778,1H-NMR (500 MHz, MeOH) spectrum showed:δ H5.41 (1H, br.s, H-12) is a hydrogen proton on a double bond,δ H3.23 (1H, t, J =2.7 Hz, H-3) is hydrogen on an oxocarbon,δ H7 methyl hydrogen protons appear between 0.75 and 1.2,δ H 2.83 (1H, dd, J=13.4, 4.4 Hz, H-18)，δ Ha plurality of CH and CH appear between 1.0 and 2.12The hydrogen signal is generated by the hydrogen signal,δ H 3.64 (2H, t, J = 13.9, 7.0 Hz), δ H3.45 (t, 2H) is a hydrogen proton on a side chain ethylenediamine group,δ H3.23 (s, 9H) is the methyl hydrogen proton on the nitrogen atom.
3. Particle size, drug loading and encapsulation efficiency of QDT-HEP-CS NPs
The particle size, polydispersity and Zeta potential of nanoparticles (QDT-HEP NPs or QDT-HEP-CS NPs) were measured using a nanometer particle size and Zeta potentiometer. The drug content of the nanoparticles is determined by high performance liquid chromatography, the total mass of the nanoparticles is measured by freeze-drying, and the drug loading rate and the encapsulation efficiency of the nanoparticles are calculated. High performance liquid phase conditions: a chromatographic column: x Select HSS T3 column (4.6 mm. times.250 mm, 5 μm); detection wavelength: 210 nm; mobile phase: acetonitrile: water = 90: 10 (V: V); flow rate: 1.0 mL-min; column temperature: 25 ℃; sample introduction amount: 20 μ L.
TABLE 1 particle size, Zeta potential, encapsulation efficiency and drug loading of QDT-HEP nanoparticles (n = 3)
| QDT: HEP | Particle size (nm) | Polymer Dispersion Index (PDI) | Zeta potential (mV) | Encapsulation efficiency (%) | Drug loading (%) |
| 4.5:4.0 | 124.30±0.10 | 0.201±0.005 | -52.87±0.67 | 50.3±0.4 | 65.8±0.5 |
| 4.5:6.0 | 168.83±2.25 | 0.192±0.008 | -45.34±2.51 | 57.7±0.8 | 47.2±0.6 |
| 4.5:8.0 | 158.23±1.29 | 0.185±0.012 | -38.48±0.49 | 63.4±0.9 | 31.1±0.5 |
| 4.5:10.0 | 185.57±4.58 | 0.208±0.009 | -44.55±2.07 | 58.4±0.3 | 27.2±0.2 |
| 4.5:12.0 | 190.91±6.43 | 0.219±0.014 | -54.88±4.38 | 54.3±1.1 | 21.1±0.4 |
TABLE 2 particle size, Zeta potential, encapsulation efficiency and drug loading of QDT-HEP nanoparticles (n = 3)
| QDT: HEP | Particle size (nm) | Polymer Dispersion Index (PDI) | Zeta potential (mV) | Encapsulation efficiency (%) | Drug loading (%) |
| 4.0:8.0 | 160.35±2.19 | 0.219±0.017 | -62.28±1.04 | 70.8±4.0 | 36.8±2.1 |
| 6.0:8.0 | 200.29±0.58 | 0.182±0.022 | -50.47±3.18 | 66.0±5.8 | 53.2±0.1 |
| 8.0:8.0 | 172.99±0.36 | 0.169±0.014 | -39.96±3.83 | 56.4±2.5 | 56.4±2.5 |
| 10.0:8.0 | 182.78±0.30 | 0.203±0.007 | -55.87±0.55 | 56.0±2.1 | 63.5±2.3 |
| 12.0:8.0 | 181.22±1.51 | 0.196±0.008 | -53.26±1.93 | 56.4±1.7 | 77.8±2.4 |
TABLE 3 particle size, Zeta potential, encapsulation efficiency and drug loading of QDT-HEP-CS nanoparticles (n = 3)
| QDT-HEP NPs:CS | Particle size (nm) | Polymer Dispersion Index (PDI) | Zeta potential (mV) | Encapsulation efficiency (%) | Drug loading (%) |
| 16:0.1 | 150.45±0.68 | 0.186±0.006 | -35.01±4.38 | 53.1±0.6 | 36.0±0.3 |
| 16:0.2 | 217.31±1.30 | 0.248±0.007 | -34.29±0.18 | 54.7±0.1 | 36.4±0.1 |
| 16:0.4 | a | a | a | a | a |
| 16:0.8 | a | a | a | a | a |
aIndicating that coagulation was observed.
4. Transmission electron microscope image
The morphology of nanoparticles of different compositions (QDT-HEP NPs or QDT-HEP-CS NPs) was observed using transmission electron microscopy. And depositing the nanoparticle solution on a copper net for 5 minutes, absorbing water by using filter paper, carrying out negative dyeing by using a 2% phosphotungstic acid solution, naturally drying at room temperature, and observing and taking a photo of a sample under a transmission electron microscope. The results are as follows:
the mass ratio of QDT-HEP to QDT-HEP-CS is 8.0: 8.0 and 8.0: 8.0: at 0.1, transmission electron microscope image analysis of the QDT nanoparticles shows that the QDT nanoparticles are all represented by helical bands with relatively uniform particle size, but the mass ratio is 8.0: 8.0: at 0.1, the nanoparticles exhibited a more compact aggregation. The particle sizes of the 2 kinds of nanoparticles under an electron microscope are all smaller than the particle size measured in water environment. This is because the weak force causes the particle surface to stretch uniformly in water, while electron microscope air drying samples causes the particles to shrink slightly. Comparing the images of the NPs, showing that the QDT and the heparin have the self-assembly behaviors driven by the non-covalent actions such as electrostatic adsorption, hydrogen bond interaction and hydrophobic interaction in the methanol aqueous solution, then the chitosan in the acetic acid aqueous solution is deposited on the surface of the QDT-HEP NPs under the electrostatic adsorption action, and the flexible helix of the QDT-HEP NPs is converted into the strip-shaped structure rigidized by the QDT-HEP-CS NPs.
5. In vitro stability test
QDT-HEP NPs and QDT-HEP-CS NPs were mixed with 1.8% NaCl, PBS (2X) and 10% glucose (glu) (1:1, v-v), mixed with RPMI1640 basal medium, fetal bovine serum +1640 complete medium, artificial gastric juice, artificial intestinal juice and rat plasma (1:4, v-v), and incubated at 37 ℃. After 24 hours, a sample was taken to measure the particle size. The results are as follows:
after the nanoparticles are incubated for 24 hours at 37 ℃, the particle size of the nanoparticles is reduced in rat serum and 1640 culture medium, and the mixed solution does not have any turbidity and precipitation phenomena, which indicates that the nanoparticles can stably exist in the liquid environment. Although incubated in different solutions, the variation in particle size among QDT-HEP-CS NPs was not significant, suggesting that NPs could be used for intravenous administration. However, the particle size increased to 390nm in 1640 medium with 10% fetal bovine serum, indicating that the nanoparticle solution could not be diluted to a range of concentrations with complete medium in cell experiments. The particle size of the nanoparticles in the artificial gastric juice and the artificial intestinal juice is increased, which shows that the QDT-HEP NPs can not exist stably in the artificial gastrointestinal juice and can not be orally taken, but the QDT-HEP-CS NPs can be prepared into an enteric preparation. The increase trend of the particle size of the nanoparticles in PBS is not obvious, but the precipitation phenomenon does not occur; the particle size in physiological saline is larger than 1000nm, which shows that the stability of the nanoparticles in PBS is general.
6. Cytotoxicity test
The cytotoxicity of the nano-sized QDT-HEP-CS NPs was determined using free-sized QDT as a control. QDT solution and QDT-HEP-CS NPs solution prepared by DMSO are diluted to final concentration of 100, 85, 70, 55, 40, 25 and 10 mu M-L by RPMI1640 basic culture medium, 4T1 cells and drugs with different concentrations are mixed according to 100 mu L-hole, each group is provided with 6 multiple holes, cells without drug treatment are taken as a normal group, and a blank control (only complete culture medium) is arranged. Incubation was performed for 12, 24, 48h after dosing. 20. mu.L of 5mg-mL thiazole blue was added, the culture was continued for 4 hours and then taken out, the supernatant in the 96-well plate was carefully removed, 150. mu.L dimethyl sulfoxide was added, the mixture was gently shaken for 10min, and the absorbance was measured at 490 nm.
Both the nano-group QDT and the free group QDT have time dependenceDependent and concentration dependent cytotoxicity, but the nano-sized QDT showed high cell viability, significantly higher than that expected from the experiment. From the inhibition rate of 4T1 cells, the inhibition effect of free QDT on 4T1 is obvious at high concentration. For the nano-group QDT-HEP-CS NPs, the inhibition effect of the drug on the tumor cells at each time is not strong. Free QDT showed significant toxicity in the first 12 hours compared to nano-sized QDT; compared to 12 hours, the prodrug nanoparticles showed higher cytotoxicity after 24 hours, and the nanoparticle toxicity was less than the free drug after 48 hours. The inhibitory effect of the nano-sized QDT on tumor cells did not change much with time compared to free-sized QDT. The inhibition effect of free group QDT on tumor cells is better than that of nano group QDT after 24 hours and 48 hours of administration, the drug effect of nano group is obviously reduced, and the cell number is obviously increased. This indicates that prodrug hydrolysis was complete in 24 hours. However, IC50Shows that the half effective dose of QDT-HEP-CS NPs at different time is lower than that of the free drug group.
7. Test of drug efficacy
4T1 mouse breast cancer cells with good growth state were taken and PBS was adjusted to 1X 107Cell concentration in mL. 0.1mL (about 1X 10) of cells at the above concentration was sampled6Individual) is inoculated to the 4 th reciprocal mammary subcutaneous fat pad on the right side of the mouse, and a tumor-bearing mouse model is established. And taking the fact that nodules can be touched on the inoculation part of the mouse on the 5 th day after the model is made as the standard for successful model making. When the mean tumor volume is as long as about 50-60mm3Dosing was started in 5 randomized groups (n = 10), model, QDT low dose, QDT-HEP-CS NPs low, medium, and high dose groups, respectively. The administration dosage of each group is determined to be 10, 20 and 40mg-kg respectively according to the equivalent dose conversion method of human and mouse. The dose was 200. mu.L per mouse, administered 7 times every other day. The model groups were given the same dose of PBS. Preparation of free QDT: the QDT powder was sonicated for 5 minutes and dissolved in 10mL of a mixed solution consisting of 0.1% tween-80, 10% absolute ethanol and PBS, and diluted one-fold with PBS before the experiment. The mice were weighed every 2 days, animals were observed and recorded for feeding, activity and death, and tumor width (W) and length (L) were recorded by digital caliper measurements according to formula V = LW2-2 calculating the tumor volume size.After 14 days of administration, blood was taken from the eyeball, the mouse was sacrificed by cervical dislocation, the tumor was peeled off, and the tumor was weighed to calculate the tumor growth inhibition rate (TGI). TGI = (1- (treatment group average tumor weight) - (control group average tumor weight)) × 100%
In situ 4T1 tumor-bearing BALB-c mice were used to evaluate the in vivo anti-tumor effect of QDT-HEP-CS NPs and the proliferation and metastasis of breast cancer. The tumor growth rate of the PBS control group was fast. Compared with the PBS control group, the tumor volume growth rates of the nano-group QDT10mg-kg, 20mg-kg and 40mg-kg are all obviously different, and the statistical significance is considered (p<0.05). The tumor volume of the nanoparticle drug treatment group is increased slower than that of the PBS control group, the growth of the tumor can be effectively inhibited, and compared with the control group, the free QDT group has no significant difference and has no statistical significance. Compared with the PBS control group, the tumor weights of the free group QDT and the nano group QDT low, medium and high dose groups are significantly different, and the statistical significance is considered (p<0.05). The mean tumor weights shown were 0.84. + -. 0.18g, 0.66. + -. 0.09g, 0.57. + -. 0.02g and 0.47. + -. 0.02g on day 15 in the free QDT and QDT-HEP-CS NPs groups, and 1.04. + -. 0.19g in the control group. The QDT-HEP-CS NPs40mg-kg group showed the least tumor, indicating that the QDT-HEP-CS NPs40mg-kg had the highest anti-tumor efficacy in all treatment groups. The tumor growth inhibitory capacity of the different groups was ranked as follows: QDT-HEP-CS NPs40mg-kg>QDT-HEP-CS NPs20mg-kg>QDT-HEP-CS NPs 10mg-kg>Free QDT10mg-kg>PBS. Compared with the PBS group, the QDT inhibition rate of the free group is about 20.25%, and the QDT-HEP- CS NPs 10, 20 and 40mg-kg show tumor inhibition rates of 36.05%, 41.67% and 53.47%, and show different tumor inhibition effects. It is shown that the nano-group QDT showed better anti-tumor effect than the free group QDT in the whole experiment.
In conclusion, the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticle prepared by the invention has good particle size and drug loading capacity, shows good stability and drug loading capacity in a rat serum medium, has the drug loading capacity of over 65 percent and the encapsulation rate of over 63 percent, and has good activity of inducing apoptosis of cancer cells and inhibiting tumor cells. The oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticle solves the problems of poor solubility and low bioavailability of oleanolic acid, can be effectively used for preparing anti-inflammatory, antihypertensive, anticancer and antibacterial medicaments, develops a new approach of the anti-inflammatory, antihypertensive, anticancer and antibacterial medicaments, is a great innovation on the anti-inflammatory, antihypertensive, anticancer and antibacterial medicaments, and has remarkable economic and social benefits.
Claims (9)
1. A preparation method of oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles is characterized by comprising the following steps:
(1) and preparation of intermediate compound:
firstly, dissolving oleanolic acid in organic solvent, and then addingN,NCarrying out ultrasonic-assisted dissolution on-diisopropylethylamine, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 1-hydroxybenzotriazole to clarify the solution, and stirring and reacting at room temperature or ice bath for 8-12 hours; then is added toN,NDimethyl ethylenediamine, performing amidation reaction at 0-25 ℃ for 12-24 hours under stirring, wherein the molar ratio of oleanolic acid to 1-ethyl-3 (3-dimethylpropylamine) carbodiimide to 1-hydroxybenzotriazole toN,NDiisopropylethylamine (diisopropylethylamine)N,N-dimethylethylenediamine (1: 1.2-3) to obtain oleanolic acid amidation intermediate compound;
the organic solvent is one or a mixture of more than two of tetrahydrofuran, dichloromethane, trichloromethane, acetone and methanol in any volume ratio;
(2) and preparing oleanolic acid quaternary ammonium salt:
dissolving the intermediate compound in an organic solvent, adding dried inorganic base and iodomethane in a molar ratio of 1: 1.5: 3-5, reacting at 50-100 ℃, and stirring in the dark for 4-12 hours to obtain oleanolic acid quaternary ammonium salt;
the inorganic base is one of potassium carbonate, sodium carbonate, cesium carbonate and sodium bicarbonate;
(3) and preparing the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution:
respectively dissolving oleanolic acid quaternary ammonium salt and heparin in methanol and deionized water, dripping the oleanolic acid quaternary ammonium salt solution into the heparin solution at room temperature, wherein the mass ratio of the oleanolic acid quaternary ammonium salt to the heparin is 4-12: 4-12, stirring overnight for 8-12h, carrying out reduced pressure rotary evaporation at 37 ℃ to obtain a film, hydrating the film with deionized water, carrying out ultrasonic treatment on the film for 5min by using a probe, centrifuging at 10000rpm for 10min, taking supernatant, and filtering with a filter membrane with the aperture of 0.45 mu m to obtain the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution;
(4) and preparing the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles:
dripping the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution into a chitosan acetic acid solution, wherein the mass ratio of the oleanolic acid quaternary ammonium salt-heparin nanoparticle to the chitosan is 16: 0.1-0.8, stirring for 2h, centrifuging for 10min at 10000rpm, taking supernatant, filtering with a filter membrane with the aperture of 0.45 mu m to obtain polyelectrolyte nanoparticle solution with surface modified chitosan, concentrating and drying to obtain the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticle;
the pH value of the chitosan acetic acid solution is 6.0.
2. The method for preparing oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles according to claim 1, comprising the steps of:
(1) and preparation of intermediate compound:
firstly, dissolving oleanolic acid in organic solvent tetrahydrofuran, and then addingN,NDiisopropylethylamine, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 1-hydroxybenzotriazole, and ultrasonic-assisted dissolution is carried out to clarify the solution, and the reaction is carried out for 10 hours under stirring at room temperature; then is added toN,NDimethyl ethylenediamine, amidating at 12 deg.c under stirring for 18 hr, and oleanolic acid/1-ethyl-3 (3-dimethylpropylamine) carbodiimide/1-hydroxybenzotriazole/N,NDiisopropylethylamine (diisopropylethylamine)N,N-dimethylethylenediamine (1: 2) to obtain an oleanolic acid amidation intermediate compound;
(2) and preparing oleanolic acid quaternary ammonium salt:
dissolving the intermediate compound in organic solvent tetrahydrofuran, adding dried inorganic base potassium carbonate and iodomethane, wherein the molar ratio of the intermediate compound to the potassium carbonate to the iodomethane is 1: 1.5: 4, reacting at 75 ℃, and stirring in the dark for 4-12 hours to obtain oleanolic acid quaternary ammonium salt;
(3) and preparing the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution:
respectively dissolving oleanolic acid quaternary ammonium salt and heparin in methanol and deionized water, dripping the oleanolic acid quaternary ammonium salt solution into the heparin solution at room temperature, wherein the mass ratio of the oleanolic acid quaternary ammonium salt to the heparin is 8: 8, stirring overnight for 10h, performing reduced pressure rotary evaporation at 37 ℃ to form a film, hydrating the film with deionized water, performing ultrasonic treatment on a probe for 5min, centrifuging at 10000rpm for 10min, taking supernatant, and filtering with a filter membrane with the aperture of 0.45 mu m to obtain the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution;
(4) and preparing the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles:
dripping the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution into a chitosan acetic acid solution, stirring for 2h, centrifuging at 10000rpm for 10min, taking supernatant, filtering with a filter membrane with the aperture of 0.45 mu m to obtain polyelectrolyte nanoparticle solution with surface modified chitosan, concentrating and drying to obtain the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticle.
3. The method for preparing oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles according to claim 1, comprising the steps of:
(1) and preparation of intermediate compound:
firstly, dissolving oleanolic acid in organic solvent methanol, and then addingN,NDiisopropylethylamine, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 1-hydroxybenzotriazole, and ultrasonic-assisted dissolution is carried out to clarify the solution, and the solution is stirred in an ice bath and reacts for 11 hours; then is added toN,NDimethyl ethylenediamine, amidating at 10 deg.c for 20 hr while stirring, and oleanolic acid/1-ethyl-3 (3-dimethylpropylamine) carbodiimide/1-hydroxybenzotriazole/N,NDiisopropylethylamine (diisopropylethylamine)N,N-dimethylethylenediamine (1: 1.3: 1)3: 1.3 to obtain the oleanolic acid amidated intermediate compound;
(2) and preparing oleanolic acid quaternary ammonium salt:
dissolving the intermediate compound in organic solvent methanol, adding dried inorganic alkali cesium carbonate and iodomethane in a molar ratio of the intermediate compound to the cesium carbonate to the iodomethane of 1: 1.5: 3, reacting at 60 ℃, and stirring in the dark for 11 hours to obtain oleanolic acid quaternary ammonium salt;
(3) and preparing the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution:
respectively dissolving oleanolic acid quaternary ammonium salt and heparin in methanol and deionized water, dripping the oleanolic acid quaternary ammonium salt solution into the heparin solution at room temperature, wherein the mass ratio of the oleanolic acid quaternary ammonium salt to the heparin is 4.5: 4, stirring overnight for 8h, carrying out reduced pressure rotary evaporation at 37 ℃ to obtain a film, hydrating the film with deionized water, carrying out ultrasonic treatment on a probe for 5min, centrifuging at 10000rpm for 10min, taking supernatant, and filtering with a filter membrane with the aperture of 0.45 mu m to obtain the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution;
(4) and preparing the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles:
dripping the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution into a chitosan acetic acid solution, stirring for 2h, centrifuging at 10000rpm for 10min, taking supernatant, filtering with a filter membrane with the aperture of 0.45 mu m to obtain polyelectrolyte nanoparticle solution with surface modified chitosan, concentrating and drying to obtain the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticle.
4. The method for preparing oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles according to claim 1, comprising the steps of:
(1) and preparation of intermediate compound:
firstly, dissolving oleanolic acid in organic solvent, and then addingN,N-Diisopropylethylamine, 1-ethyl-3 (3-dimethylpropylamine) carbodiimide and 1-hydroxybenzotriazole, and ultrasonic-assisted dissolution is carried out to clarify the solution, and the solution is stirred and reacts for 9 hours at room temperature; then is added toN,NDimethyl ethylenediamine, amidating at 23 deg.c with stirring for 13 hr, and oleanolic acid/1-ethyl-3 (3-dimethylpropylamine) carbodiimide/1-hydroxybenzotriazole/N,N-Diisopropylethylamine: diisopropylethylamineN,N-dimethylethylenediamine (1: 2.9) to obtain oleanolic acid amidation intermediate compound;
the organic solvent is a mixture of dichloromethane: trichloromethane = 1:1 in volume ratio;
(2) and preparing oleanolic acid quaternary ammonium salt:
dissolving the intermediate compound in an organic solvent, adding dried inorganic alkali sodium bicarbonate and iodomethane in a molar ratio of the intermediate compound to the sodium bicarbonate to the iodomethane of 1: 1.5: 5, reacting at 90 ℃, and stirring in the dark for 5 hours to obtain oleanolic acid quaternary ammonium salt;
(3) and preparing the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution:
respectively dissolving oleanolic acid quaternary ammonium salt and heparin in methanol and deionized water, dripping the oleanolic acid quaternary ammonium salt solution into the heparin solution at room temperature, wherein the mass ratio of the oleanolic acid quaternary ammonium salt to the heparin is 6: 8, stirring overnight for 12h, performing reduced pressure rotary evaporation at 37 ℃ to form a film, hydrating the film with deionized water, performing ultrasonic treatment on a probe for 5min, centrifuging at 10000rpm for 10min, taking supernatant, and filtering with a filter membrane with the aperture of 0.45 mu m to obtain the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution;
(4) and preparing oleanolic acid quaternary ammonium salt-heparin chitosan nanoparticles:
dripping the oleanolic acid quaternary ammonium salt-heparin nanoparticle solution into a chitosan acetic acid solution, stirring for 2h, centrifuging at 10000rpm for 10min, taking supernatant, filtering with a filter membrane with the aperture of 0.45 mu m to obtain polyelectrolyte nanoparticle solution with surface modified chitosan, concentrating and drying to obtain the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticle.
5. The method for preparing oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles according to claim 1, which comprisesIs characterized in that the preparation of the intermediate compound comprises weighing 200mg of oleanolic acid, 180mg of HOBT and 250mg of EDCI into a single-mouth bottle, and dropwise adding 200 mu L of DIPEA and CH2Cl25mL of the solution is ultrasonically dissolved, and the solution is stirred for 8 hours in ice-water bath; monitoring the reaction process by thin layer chromatography, developing agent: dichloromethane: methanol: 15: 1, developer: the method comprises the following steps of (1) dropwise adding 130 mu L of N, N-dimethylethylenediamine when a raw material point disappears, reacting at room temperature, stopping the reaction when an activation point disappears, carrying out reduced pressure rotary evaporation to remove a solvent, dissolving a crude product by using ethyl acetate, extracting for 3 times by using an isometric saturated NaCl aqueous solution, concentrating by using a rotary evaporator to obtain a white oily substance, separating and purifying by using a flash chromatography system by using petroleum ether/ethyl acetate (volume ratio) of 5: 1, evaporating the solvent by using a centrifugal concentrator, and carrying out vacuum drying to obtain a white solid oleanolic acid amidated intermediate compound.
6. The method for preparing oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticle as claimed in claim 1, wherein the preparation of oleanolic acid quaternary ammonium salt comprises precisely weighing 100mg of oleanolic acid amidated intermediate compound, dissolving with 2mL of MeOH, adding K2CO331.5mg, then adding 35.5 mu L of methyl iodide dropwise, refluxing at 70 ℃, monitoring the reaction process by thin layer chromatography, developing agent: dichloromethane: methanol 5: 1, developer: stopping the reaction when the mass concentration of the sulfuric acid-ethanol is 10 percent and the raw material point disappears; filtering the reaction solution, and removing the solvent by reduced pressure evaporation to obtain a white oily substance; separating and purifying with acetonitrile/pure water at a volume ratio of 90: 10, volatilizing solvent in a test tube concentrator, recrystallizing with diethyl ether, and vacuum drying to obtain white solid quaternary ammonium oleanolic acid salt.
7. The method for preparing the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticle according to claim 1, wherein the mass ratio of oleanolic acid quaternary ammonium salt to heparin is further 4.5: 6, 4.5: 8, 4.5: 10, 4.5: 12; 4: 8, 10: 8 or 12: 8.
8. The method for preparing the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticle according to claim 1, wherein the mass ratio of the oleanolic acid quaternary ammonium salt-heparin nanoparticle to the chitosan is further 16: 0.7 or 16: 0.8.
9. Use of the oleanolic acid quaternary ammonium salt-heparin-chitosan nanoparticles prepared by the method of claims 1-8 in preparing anti-inflammatory, antihypertensive, anticancer and antibacterial drugs.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004098564A2 (en) * | 2003-05-02 | 2004-11-18 | The Board Of Trustees Of The University Of Illinois | Biodegradable nanoparticles comprising an aminoglycoside and a polymer like a polysaccharide |
| CN102813937A (en) * | 2012-06-12 | 2012-12-12 | 天津大学 | Hydrophobic drug containing polyelectrolyte complex, its preparation method and application thereof |
| CN102988999A (en) * | 2012-05-09 | 2013-03-27 | 中国药科大学 | Curcumin-polysaccharide conjugate as well as preparation method and application thereof |
| US20190000763A1 (en) * | 2015-08-06 | 2019-01-03 | Pratibha Pilgaonkar | Particulate delivery systems |
| CN109662956A (en) * | 2018-11-15 | 2019-04-23 | 东华大学 | A kind of application of the chitosan drug-loading nano particle of oleanolic acid grafting |
-
2021
- 2021-04-13 CN CN202110393056.8A patent/CN113069554B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2004098564A2 (en) * | 2003-05-02 | 2004-11-18 | The Board Of Trustees Of The University Of Illinois | Biodegradable nanoparticles comprising an aminoglycoside and a polymer like a polysaccharide |
| CN102988999A (en) * | 2012-05-09 | 2013-03-27 | 中国药科大学 | Curcumin-polysaccharide conjugate as well as preparation method and application thereof |
| CN102813937A (en) * | 2012-06-12 | 2012-12-12 | 天津大学 | Hydrophobic drug containing polyelectrolyte complex, its preparation method and application thereof |
| US20190000763A1 (en) * | 2015-08-06 | 2019-01-03 | Pratibha Pilgaonkar | Particulate delivery systems |
| CN109662956A (en) * | 2018-11-15 | 2019-04-23 | 东华大学 | A kind of application of the chitosan drug-loading nano particle of oleanolic acid grafting |
Non-Patent Citations (2)
| Title |
|---|
| MIDHUN B THOMAS 等: ""Intracellular delivery of doxorubicin encapsulated in novel pH-responsive chitosan/heparin nanocapsules"", 《INTERNATIONAL JOURNAL OF NANOMEDICINE》 * |
| 杜建红 等: ""羟丙基壳聚糖/肝素纳米载药体系的制备、释放及抗肿瘤特性分析"", 《中国组织工程研究》 * |
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